Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone–stimulated Cl^– transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR_inh-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K_I approximately 300 nM. CFTR_inh-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR_inh-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl^– channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K⁺ channels, or a series of other transporters. A single intraperitoneal injection of CFTR_inh-172 (250 μg/kg) in mice reduced by more than 90% cholera toxin–induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.